The nicotinic acetylcholine receptor (AChR) will undoubtedly be the first ligand-gated channel whose architecture, gating mechanism, mode of ion permeation and multiple modes of inhibition will be understood at the atomic level. Present models of this prototypic ion channel explain elegantly a large body of data and yet, they are incomplete and often contradictory. Much has been learnt about this channel from studies of noncompetitive inhibitors (NCI's) that bind to a site within the channel itself. In the present project, this site will be further studied by a combination of a comparative approach with genetic manipulations and structure-function studies. Two receptor subtypes, AChR from Torpedo electric organ and from mouse muscle, are known to have different affinity for positively-charged NCI's but not for the neutral cembranoids (new NCI's discovered in the P.I.'s laboratory; Eterovic et al., 1993a,b). Hybrid receptors, constructed from Torpedo and mouse subunits, will be expressed in Xenopus laevis oocytes and studied with a voltage.clamp. These experiments will indicate which subunit(s) determines the affinity for NCI's. Chimeric receptors will be constructed next in which Torpedo M2 segments will be exchanged for their counterparts in the mouse receptor and vice versa. Finally, specific amino acids in the M2 segments will be changed by site- directed mutagenesis to determine which residues interact with each type of inhibitors (cationic versus neutral). The topology of the cembranoids binding site will be further studied through structure-function relationships for a series of natural cembranoid analogues. These will be tested on oocyte-expressed Torpedo receptor and in binding studies with AChR-rich membranes. In addition to the aesthetic appeal of pure knowledge, a detailed understanding of the AChR ion channel would make possible to design a 'perfect' muscle relaxant, potent yet devoid of side effects. This project is well suited for student training, since it addresses an important scientific problem and offers a diversity of modern and classical experimental techniques and approaches.